Diversité et origine génétique des espèces de la sous famille des Aurantioideae et décryptage des structures interspécifiques des génomes des agrumes modernes

L'étude de l'espaceur intergénique tmL-tmF chez les espèces tunisiennes du genre Citrus montre la présence d'une seule copie du pseudogène tmF chez toutes les variétés analysées. Les valeurs positives et non significatives des tests de neutralité plaident en faveur d'un modèle d'évolution neutre et supportent l'hypothèse d'un scénario démographique stable. Nos résultats montrent clairement la contribution des pools génétiques de C. reticulata et C. maxima dans le développement des espèces secondaires tunisiennes. L'analyse basée sur les séquences nucléotidiques de huit régions génomiques chloroplastiques de 79 variétés de la sous famille des Aurantioideae révèle, via les marqueurs SNPs, une différenciation taxonomique au niveau des tribus, des soustribus, des genres et des espèces. 166 SNPs diagnostiques des 54 clades analysés ont été identifiés suivis d'une sélection de 40 KASPars. L'application de ces marqueurs chez 108 variétés montre un haut taux de transférabilité au sein de la sous famille des Aurantioideae et une cohérence avec les analyses génétiques antérieures du génome chloroplastique 21 clés marqueurs de clade catégoriquement diagnostique de 19 clades ont été identifiées. L'analyse GBS des 55 variétés d'agrumes par séquençage ILLUMINA H1SEQ2000 s'avère efficace dans d'identification des polymorphismes diagnostiques (12564 SNPs/lnDels) de la différenciation C. reticulata/C. maxima couvrant l'ensemble du génome nécessaire au déchiffrage de l'origine phylogénomique tout au long des neuf chromosomes des 55 variétés. L'approche adoptée confirme les analyses récentes basées sur des données de séquence de génome complet pour la clémentine, l'orange douce et amère et la mandarine 'Ponkan'. La technique GBS couplée à la détection des points polymorphes diagnostiques s'avère très efficace dans le décryptage des caryotypes phylogénomiques des variétés qui dérivent d'une mosaïque de deux espèces ancestrales. Le mélange C. reticulata/C. maxima doit être l'élément majeur de la variabilité phénotypique révélée élevée de ces ressources. (Résumé d'auteur

Details of antibodies used in the study.

<p>Details of antibodies used in the study.</p

Effect of mitofusin deficiency on respiratory complexes protein level in MEF cells.

<p>The cells were grown in the medium supplemented with FBS. Selected respiratory complexes proteins were detected in MEFwt and MEF^Mfn2-/- cells by Western blotting. Representative blot of selected mitochondrial complexes subunits and densitometric analysis of at least six independent experiments are shown. Data are presented as mean immunoreactivity normalized to immunoreactivity of PCNA ± S.D. Immunoreactivity of these proteins in wild type cells is assumed as 100%, n = 6–8, *p<0.05.</p

Effect of mitofusin 2 deficiency on ATP content in MEF cells treated with oligomycin and iodoacetate.

<p>MEFwt and MEF^Mfn2-/- cells grown in the medium supplemented with FBS were rinsed and incubated for 10 min in the presence of 1 mM pyruvate and 5.6 mM glucose (A) or in glucose-free Krebs-Henseleit solution (B). ATP was measured in neutralized acidic cell extracts. Where indicated the cells were treated with oligomycin (0.1 μg/ml) and iodoacetate (1 mM) to inhibit oxidative phosphorylation and glycolysis, respectively. Data are expressed as a mean amount of ATP (nanomoles per milligram of protein) ± S.D.; n = 3–5 * p < 0.05.</p

Effect of mitofusin deficiency on the amount of TOM20 protein and mitochondrial mass in MEF cells.

<p>The cells were grown in the medium supplemented with FBS. (A). Relative amount of mitochondrial marker TOM20 in MEFwt and MEF^Mfn2-/- cells were tested immunochemically by Western blotting and normalized to immunoreactivity of PCNA. Representative blot as well as mean values of immunoreactivity ± S.D. for TOM20 are shown. Immunoreactivity of this protein in wild type cells is assumed as 100%, n = 4, **p<0.001. (B) Comparison of three loading markers (PCNA, β-actin and GAPDH) level in MEFwt and MEF^Mfn2-/- cells. There are no differences in the immunoreactivity between these proteins in both cell lines tested. (C) Mitochondrial mass was estimated by NAO staining of cardiolipin. Fluorescence was measured with flow cytometry. n = 6, *** p < 0.0005.</p

Respiratory capacity of MEFwt and MEF^Mfn2-/- cells.

<p>The cells were grown in media supplemented with either FBS or BCS, as indicated. Respiratory capacity was expressed as a ratio between maximal (upon addition of CCCP) and minimal (in the presence of oligomycin prior to addition CCCP) rates of oxygen consumption. Results represent mean values of the ratio ± S.D. n = 4.</p><p>Respiratory capacity of MEFwt and MEF^Mfn2-/- cells.</p

Effect of mitofusin deficiency on the amount of TFAM and PGC-1α protein in MEF cells.

<p>The cells were grown in the medium supplemented with FBS. Relative amounts of both proteins in MEFwt and MEF^Mfn2-/- cells were tested immunochemically by Western blotting and normalized to immunoreactivity of PCNA. Representative blots and mean values of immunoreactivity ± S.D. are shown. Immunoreactivity of these proteins in wild type cells is assumed as 100%, n = 4, *p<0.01. ** p<0.05.</p

Relative effects of oligomycin and iodoacetate on ATP content in MEF cells incubated with or without glucose in the reaction buffer.

<p>The cells were grown in the medium supplemented with FBS. Data shown in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134162#pone.0134162.g008" target="_blank">Fig 8</a> are presented as ratios between amount of ATP in cells incubated with oligomycin (olig) or iodoacetate (IA) and untreated cells of the same type (MEFwt_, or MEF^Mfn2-/-, respectively). Amount of ATP in cells untreated with oligomycin or CCCP was assumed to be 1. Results represent mean values of the ratio ± S.D. for n = 3–5. p values were calculated using paired <i>t</i>-Student test in a relation to ATP formation rate in cells not treated with inhibitors.</p><p>Relative effects of oligomycin and iodoacetate on ATP content in MEF cells incubated with or without glucose in the reaction buffer.</p

Oxygen consumption by MEFwt and MEF^Mfn2-/- cells.

<p>The cells were grown in media supplemented with either FBS or BCS. Oxygen consumption was measured polarographically at 37°C in a substrate free buffered saline solution (PBS with Mg²⁺ and Ca²⁺), and after sequential administration of 1 mM pyruvate, 5 mM glucose, 0.1 mg/ml oligomycin and 1 μM CCCP. Data are expressed as pmoles O₂ x s^-1/mg protein and show mean values ± S.D. n = 4; *p<0.01, **p<0.001, ***p<0.0001.</p

Mitochondrial membrane potential in MEFwt and MEF^Mfn2-/- cells.

<p>The cells were grown in the medium supplemented with FBS. Mitochondrial membrane potential was determined fluorimetrically with JC-1 probe using flow cytometry. JC-1 fluorescence was measured in TO-PRO-3 negative cells (more than 95% of total population), which were assumed as 100%. Ordinate shows a proportion of cells with energized mitochondria. Data show mean values ± S.D. n = 6.</p